Preparation of trichloro-s-triazine activated supports for coupling ligands

ABSTRACT

Proteins and non-protein affinity ligands are covalently bonded to trichloro-s-triazine activated supports. The activated support is prepared by reacting a water-free insoluble solid support with trichloro-s-triazine in a non-aqueous medium and neutralizing HCl generated during the reaction with a tertiary amine which does not form an insoluble complex with the trichloro-s-triazine.

The invention described herein was made in the course of work under agrant or award from the Department of Health, Education and Welfare.

BACKGROUND AND SUMMARY OF THE INVENTION

The present invention is concerned with new methods and reagents forcoupling proteins and a wide variety of non-protein affinity ligands bystrong covalent chemical bonds to solid-phase supports for thepreparation of solid-phase catalysts and biospecific adsorbents.

Biospecific adsorbents have become very important tools for theisolation of biologic macromolecules, while immobilized enzymes havefound wide use as durable, solid-phase catalysts. Such materials areprepared by chemically linking enzymes, inhibitors, or other biospecificcompounds to a solid-phase support. Various methods and reagents havebeen described for the purpose of coupling, but each has its peculiardrawbacks, including: (a) weak chemical bonding; (b) ionic bonds; (c)necessity for strong conditions such as high temperature and pH to drivethe reaction; (d) ride reactions; and (e) limited range of chemicalgroups with which the coupling reagent can react.

Previously coupling reagents include the reagent most widely utilized asa coupler, cyanogen bromide, as described by Jacoby et al. in MethodsEnzymol. 34, 1974. This reagent is reacted with support such as agaroseand cellulose in strongly alkaline solution where numerous sidereactions occur. The cyanogen bromide-activated support subsequentlyreacts poorly with nucleophiles other than alkylamines, a severelimitation on the kinds of enzymes, ligands and other compounds whichcan be coupled. The coupling bonds, probably isourea bonds, are onlymoderately stable, and substantial amounts of the bound substance may"bleed" under practical conditions. This instability limits the usefullife of the adsorbent or solid-phase catalyst and may add undesirable orpotentially toxic contaminants to the product. Moreover, these isoureabonds are positively charged at neutral pH and can cause non-specificadsorption.

In contrast, reagents of a type that can provide strong covalent bondsbetween diverse supports and various nucleophilic groups of ligandsunder mild conditions have been employed in the dye industry since theintroduction of "reactive" dyes. The most versatile and widely employedof these coupling reagents is trichloro-s-triazine (TsT). This reagentcontains three reactive chlorines, the first of which is displaced togive a substituted dichloro-s-triazine (DsT), and the second to give adisubstituted monochloro-s-triazine (MsT). Conditions for controlledsubstitutions by various nucleophiles have received comparatively littlestudy.

Like cyanogen bromide, TsT has been reacted with supports in stronglyalkaline aqueous media where hydrolytic side reactions predominate. Inthe procedure as described by Kay et al. in Nature 217:641, (1968), theamount of TsT which reacts with support and that which hydrolyzes arecompetitive functions of temperature, pH and reagent concentration, andthe extent of activation of the support cannot be predicted accurately.Moreover, the activated support is also subject to hydrolysis. Recently,Kay and Lilly have introduced the less reactive coupler,2-amino-4,6-dichloro-s-triazine, as discussed in Biochim. Biophys. Acta.198:276 (1970). However, in alkaline aqueous media, this reagent issubject to the same drawbacks as TsT during the activation reaction and,in addition, vigorous conditions are required for coupling the ligands.The major problems with such prior art methods are: (1) couplers areused empirically as others have used cyanogen bromide; (2) althoughtriazine coupling provides strong bonds between support and proteins, noattention has been previously paid to competitive hydrolytic sidereactions and to the introduction of adsorptive ionic sites; (3) theamount of ligand incorporated cannot be accurately controlled orpredicted; and (4) the procedures are not satisfactory for theincorporation of small ligands in organic phase.

In order to circumvent the problem of TsT hydrolysis and to performstep-wise reactions at individual chlorines of TsT, by the presentinvention there have been developed methods for reaction in non-aqueousmedia. The critical conditions were found to include appropriate polarorganic solvents and suitable organic bases to neutralize the HClgenerated. With polyol supports like cellulose or cross-linked agarose,as described by Porath et al. in J. Chromatogr. 60:167 (1971), reactionswith TsT have been found to occur smoothly and predictably in organicphase to give a (DsT)-dichloro-s-triazine substituted support. Thisactivated support could be reacted in organic phase at one or bothremaining chlorines, depending upon the nucleophiles involved and thereaction conditions.

It has also been found, in accordance with the present invention, thatweak nucleophiles such as aniline react at room temperature in organicsolvent with one of the chlorines of the DsT-support, leaving a singlesite for subsequent reaction. In fact, the initial "activation" reactioncould be well controlled without side reactions to give a DsT-support;and a subsequent reaction with a weak nucleophile could be performedwithout side reactions to give an MsT-support. Finally, the singleremaining chlorine of the MsT-support is less reactive than theDsT-support or TsT and is less susceptible to hydrolysis at moderate pHand temperature. Therefore, it can be reacted with nucleophiles inorganic or in aqueous solution under mild conditions of pH andtemperature to give the desired products.

In J. Biol. Chem. 252, 3578 (1977) Abuchowski et al. have reported thecovalent attachment of polyethylene glycol (PEG) to proteins using TsTas a linking agent. While the coupling of an aliphatic alcohol to TsT inorganic phase is the initial reaction in both the Abuchowski et al.procedure and in the present invention, this initial coupling is theentire extent of any similarity.

In their initial coupling reaction, Abuchowski et al. use benzene as asolvent for both PEG and TsT and use Na₂ CO₃ to neutralize the HClgenerated. This method, while adequate only for the coupling of TsT andPEG in solution phase, is not satisfactory for the coupling of TsT andany of the solid-phase supports utilized in affinity preparations, suchas Sepharose, cellulose or polyvinyl alcohol. Benzene has proved to be apoor medium for reactions involving Sepharose and other hydrophilicpolymers, most likely because of the non-polar nature of benzene. Amajor problem in the case of Sepharose or other polymers initially inaqueous phase is the transfer to benezene. It has also been observedthat benzene seems to have an adverse effect on Sepharose structure.Furthermore, benzene is not a particularly good solvent for TsT, asdiscussed in a "s-Triazines and Derivatives," in The Chemistry ofHeterocyclic Compounds (E. M. Smolin and O. Rapoport, eds.).Interscience Publishers, Inc., New York, Vol. 13, 1959.

Neutralization of the HCl generated is inefficient when the base isinsoluble, as is the case of Na₂ CO₃ in benzene. Inefficientneutralization of the HCl could permit conversion of the alcohol to thecorresponding alkyl chloride, a side reaction known to occur under theseconditions. Also, incorporation of chlorine into the Sepharose matrix islikely to impart undesirable properties.

By the present invention, there has been achieved the ability tocircumvent many of the problems inherent in the Abuchowski et al.procedure, by conducting the initial activation reaction in dioxane orother organic solvents and by neutralizing the HCl generated with asoluble organic base such as N,N-diisopropylethylamine or other tertiaryamine which does not form an insoluble complex with TsT in organicphase. Of the several reaction media which have been tried, dioxane wasfound to be best as it is both compatible with Sepharose and otherhydrophilic polymers and is also a superior solvent for TsT. The use ofa soluble organic base is also novel, and selection of the correct baseis critical as most form insoluble complexes with TsT.

After the initial activation step, the two procedures are obviouslydifferent. Abuchowski et al. are satisifed with empirical coupling ofdichloro-s-triazine-PEG to proteins in aqueous phase under alkalineconditions. The reference also states that a considerable excess ofactivated PEG must be used because hydrolysis of the second chlorine ofthe triazine occurs readily. This is a common feature of triazinereactions in aqueous or mixed aqueous-organic phase and it preventsaccurate control and predictability of the reaction.

Unreacted and hydrolyzed PEG-dichloro-s-triazine are, of course, solubleand and separable from the PEG-coupled protein. This is not the case,however, if one wants a broader objective, i.e., coupling of aninsoluble solid-phase compound such as cellulose-dichloro-s-triazine toa ligand where any side reactions such as hydrolysis affect the desiredproduct. In the procedure of the present invention, control isestablished by replacing the second triazine chloride by a weaknucleophile such as aniline. This leaves a single chlorine available forreaction with nucleophilic groups of the proteins or other substances tobe immobilized. This single chlorine can be replaced quantitatively byaliphatic amines under mold conditions, yet it does not hydrolyzereadily below pH 9 at room temperature.

Various supports (polyols) can be reacted with TsT in accordance withthe present invention, but all such supports must be rid of water priorto the initial reaction. Where suitable, drying can be performed byheating in vacuo, or water may be displaced from the support by washingwith dry, miscible organic solvents such as 1,4-dioxane, acetonitrileand similar solvents. In addition, the parameters of the reactionconditions can be altered to effect changes in the amount of TsT whichreacts with the support, including changes in reaction temperatures,reagent concentration, and the duration of the reaction.

The reaction of TsT with the support in organic phase requires thepresence of a suitable organic base. The bases generally employed inanalogous acyl chloride type reactions such as triethylamine, pyridine,N-ethyl morpholine, and lutidine, were found to form insoluble complexeswith TsT in organic solvents. However, it was found that tertiary aminessuch as N,N-dimethylaniline and N,N-diisopropylethylamine did not formsuch complexes and performed satisfactorily.

BRIEF DESCRIPTION OF THE DRAWINGS

The advantages and features of the present invention will be more fullyunderstood from the following description of the preferred embodiments,taken in conjunction with the accompanying drawings, wherein:

FIG. 1 is a graph showing a comparison of the stability of ligandsattaches with CNBr to those attached with TsT; and

FIG. 2 is a graph showing various characteristics of protein coupled toTsT-activated resin.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following is a general procedure for the preparation of thereactions of triazine-linked bioaffinity resins beginning with anaqueous suspension of support such as cross-linked agarose. Thisprocedure is set forth schematically as follows: ##STR1##

An aqueous suspension of the support is washed with a series ofdioxane-water solutions in which the amount of dioxane increases from 0to 100%. The support is transferred to a round bottom flask equippedwith a glass-sealed stirrer and water condenser. Transfer isaccomplished with a small amount of dioxane. The reaction flask isimmersed in a thermostated oil bath and its contents stirred at lowspeed. N,N-diisopropylethylamine or N,N-dimethylaniline in dioxane isadded and the reaction is allowed to equilibrate. The reaction isinitiated by the addition of trichloro-s-triazine dissolved in dioxane.Generally, fluid and solid phases are of equal volume, and baseconcentration is twice that of the trichloro-s-triazine. The amount oftriazine incorporated into the support resin depends ontrichloro-s-triazine concentration, length of reaction and reactiontemperature.

The activated DsT support is then washed with several bed volumes ofdioxane. At this point the activated support can be reacted in severaldifferent ways: (1) it can be reacted in organic dioxane with a strongnucleophile such as an alkyl amine to give a dialkyl-triazine-resin; (2)it can be reacted in dioxane with a weak nucleophile such as an arylamine to give a monoaryl-triazine-resin with a chlorine still availablefor further reaction; (3) the monoaryl-triazine-resin can be washed withdioxane-water solutions containing decreasing amounts of dioxane and theresin, now in aqueous solution, can be reacted with either proteins orother nucleophiles; or (4) the organic solvent can be removed from themonoarylmonochloro-s-triazine-resin by freeze-drying or other gentlemeans and the dried, activated resin stored for later use.

EXAMPLE 1

As a specific example, 100 ml of Sepharose CL-4B was washed with 200 mlof a mixture of water:dioxane (30:70) followed by 200 ml water:dioxane(70:30) and finally by 1000 ml dioxane. Washing was accomplished on asintered glass funnel under vacuum. The resin was allowed to standovernight in a glass stoppered graduated cylinder to accurately measurethe bed volume and, after removing the excess dioxane, the settled gelwas transferred with 60 ml dioxane to a 500 ml 3-necked round bottomflask equipped with a water jacketed condenser and a glass-sealedstirrer. The reaction flask was immersed in a thermostated oil bathmaintained at 50°±2° C. and the contents were stirred at 100 rpm. Theamount of 20 ml of 2 M N,N-diisopropylethylamine in dioxane was thenadded. After 30 min, 20 ml of 1 M trichloro-s-triazine in dioxane wasadded to initiate the reaction. After 60 min at 50°, the activated resinwas washed on a sintered glass funnel with 1000 ml of dioxane. The resinwas found to contain 112 μmoles triazine per gram of resin. A portion ofthe resin was then reacted with 2 volumes per bed volume of resin, of 1M ethylene diamine in dioxane, for 30 min, at room temperature and wasfound to couple 244 μmoles of diamine per gram of resin.

A second portion was reacted with 2 volumes of aniline in dioxane atroom temperature for 30 min. and was subsequently washed with 5 bedvolumes of dioxane on a sintered glass funnel. A portion of this latterresin was reacted with 2 bed volumes of 1 M ethylenediamine in dioxaneat room temperature for 30 min and was found to couple 112 μmoles ofdiamine per gram of resin. A second portion of the aniline-treated resinwas washed with 2 bed volumes of water:dioxane (70:30) at 4° C. andfinally by 10 bed volumes of water at 4° C. on a sintered glass funnel.

A portion of the resin in aqueous phase was incubated with 2 bed volumesof 0.66 M epsilon amino caproic acid, 0.30 M sodium borate, 0.30 Msodium chloride buffer, pH 8.5, for 22 hours at room temperature, afterwhich time 116 μmoles of 6-aminohexanoic acid was incorporated into theresin. Another portion of the resin in aqueous phase was reacted with 4bed volumes of a solution of bovine serum albumin, 10 mg/ml in 0.30 Msodium borate, 0.30 M sodium chloride buffer, pH 8.0, for 22 hours atroom temperature. This resin was found to have coupled 42.3 mg ofprotein per gram of resin.

EXAMPLE 2

To compare the stability of ligands attached to agarose with CNBr tothose attached with TsT, ¹⁴ C-bovine serum albumin (BSA) and ¹⁴ C-EACAwere each coupled to Sepharose CL-6B using these two coupling reagents.The resins were then treated under conditions which would acceleratehydrolysis of the coupling bonds, namely elevated temperature and pH asshown in FIG. 1. The graph of FIG. 1 shows the results of hydrolysis of¹⁴ C-EACA-Sepharose and ¹⁴ C-BSA-Sepharose in 0.33 M Na₂ CO₃, pH 11.2 at50° C. Packed resin was suspended in buffer and at the indicatedintervals aliquots were removed and assayed for radioactivity. Totalradioactivity represents the total amount of ligand bound to the resin.¹⁴ C-EACA and ¹⁴ C-BSA alkylated with ¹⁴ C-iodoacetamide and containing0.6 mole carboxyamidomethyl groups per mole of protein (1.8×10⁵ cpm/mg)prepared in accordance with procedures set forth, for example, by Finlayet al., J. Biol. Chem. 245, 5258 (1970), were coupled to CNBr-activatedSepharose CL-6B in 0.3 M Na Borate, 0.3 M NaCl, pH 8.0, by the procedureoutlined by Parikh et al. in Methods Enzymol. 34:77 (1974). TheBSA-Sepharose linked with CNBr contained 35.3 mg of protein/g resin andthe EACA-Sepharose contained 317 μmoles of amine/g resin. ¹⁴ C-BSA and¹⁴ C-EACA were coupled to triazine-activated Sepharose CL-6B in 0.22 MNa borate, 0.22 M NaCl, pH 8.0. The BSA-Sepharose prepared by triazineactivation contained 42.3 mg of protein/g resin and the EACA-Sepharosecontained 140 μmoles of amine/g resin. Packed resin, 1 ml, was suspendedin 2.0 ml 0.5 M Na₂ CO₃, pH 11.2, at 50°. At the intervals indicated inFIG. 1, the resin suspension was centrifuged and an aliquot from thesupernatant fraction removed and counted. The data clearly show thetriazine linkage to be superior to the CNBr linkage for both proteinsand small molecule ligands.

Referring to FIG. 2, this graph shows that the amount of protein coupledto triazine activated resin is dependent on the amount of free proteinpresent in the coupling reaction.

The data of FIG. 2 is concerned with the effect of coupling of bovineserum albumin to Sepharose CL-MsT. Samples of Sepharose CL-6BMsT (100mg), lyophilized from dioxane, were suspended in 5.0 ml of 0.15 M NaCl,0.1 M NaBorate, pH 8.0. After mixing at 0° for 1 min, the suspensionswere centrifuged and the supernatants aspirated to yield a total volumeof resin plus liquid of 1.5 ml. ¹⁴ C-CM-Bovine serum albumin in 1.0 mlof this same buffer was then added and the reactions were incubated withrocking at room temperature. After 44 hrs, the resins were washed with1.0 M Tris-HCl, pH 8.8, water and 50% ethanol and then lyophilized. Theamount of protein coupled was calculated after counting of weighedaliquots of the dried resin. It is interesting to note that with thisparticular lot of resin, 125 mg of albumin was coupled per gram with noindication that the resin had become saturated.

In Table I it is shown that in the pH range of 6-9, temperature has agreater effect on coupling efficiency than does pH. In preparing theinformation as shown in Table I, samples of Sepharose CL-MsT (100 mg)lyophilized from dioxane, were suspended in 5.0 ml buffer (pH 6 and7:0.1 M NaPhosphate; pH 8 and 9:0.1 M NaBorate) containing 0.15 M NaCl.After mixing at 0° C. for 1 min, the suspensions were centrifuged andthe supernatants aspirated to yield a total volume of resin plus liquidof 1.9 ml. ¹⁴ C-CM bovine serum albumin (0.1 ml, 9.98 mg, 1.17×10⁵ cpm)was added and the reactions were incubated with rocking for 24 hrs.Samples were washed and counted as described in connection with FIG. 1.

                  TABLE I                                                         ______________________________________                                        Coupling of                                                                   Bovine Serum Albumin to Sepharose CL -MsT;                                    Effect of temperature and pH                                                                          mg protein coupled                                    Temperature    pH       g resin                                               ______________________________________                                                       6        19.7                                                                 7        19.8                                                   8°                                                                                   8        20.7                                                                 9        21.5                                                                 6        28.0                                                                 7        26.2                                                  27°                                                                                   8        30.2                                                                 9        33.3                                                                 6        36.1                                                                 7        45.9                                                  45°                                                                                   8        51.1                                                                 9        48.9                                                  ______________________________________                                    

In Tables II and III there are listed some of the enzymes, otherproteins and other substances which have been coupled to TsT-activatedSepharose. Of the enzymes which have been coupled in accordance with thepresent invention, the one with the greatest commercial value isprobably bacterial α-amylase which converts starch to dextrins and isused in the paper and textile industries and in the production ofglucose.

In preparing the information as shown in Table II, reaction mixturescontained 100 mg of activated resin in a total volume of 2.0 ml 0.1 NaBorate, 0.15 M NaCl, pH 8.0 at the protein concentration indicated thefirst column of Table II. Reactions were conducted at 25°±2° C. withgentle rocking for 24 hours. The resins were then washed by filtrationfirst with 1.0 M tris-Cl, pH 8.8 followed by 0.05 M tris-Cl, 0.15 MNaCl, pH 8.3. Resins were assayed for protein and enzymic activity asdescribed previously.

It is thought that the invention and many of its attendant advantageswill be understood from the foregoing description, and it will beapparent that various changes may be made in the methods as describedherein without departing from the spirit and scope of the invention orsacrificing its material advantages, the forms hereinbefore describedbeing merely preferred embodiments thereof.

                                      TABLE II                                    __________________________________________________________________________    COUPLING OF ENZYMES TO                                                        Sepharose CL-6B WITH TsT IN AQUEOUS PHASE AT pH 8                                                         ENZYME MG/G                                                  COUPLING   %     COUPLED                                                                              RESIN                                                 CONCENTRATION                                                                            PROTEIN                                                                             AS     AS     %                                   ENZYME     (MG/ML)    BOUND PROTEIN                                                                              ACTIVITY                                                                             ACTIVITY                            __________________________________________________________________________    δ-Amylase                                                                          3.9        13.2  10.2   6.2    60.2                                Lactic Dehydrogenase                                                                     3.7        48.9  36.2   3.3    9.1                                 Cellulase  4.5        8.3   7.5    1.2    16.0                                Trypsin    4.3        35.9  30.9   3.6A   11.7                                Chymotrypsin                                                                             4.3        36.2  31.1   2.9    9.3                                 __________________________________________________________________________

                  TABLE III                                                       ______________________________________                                        LIGANDS ATTACHED TO CL-SEPHAROSE                                              WITH TsT IN AQUEOUS PHASE                                                             Amount                                                                        Coupled/g                                                             Ligand  Resin     Biological Acitivity                                        ______________________________________                                        Fibrinogen                                                                            28-142 mg As fibrin-Sepharose, adsorbs                                                  24-130 mg fibrinogen per G resin                            Anti-                                                                         fibrinogen                                                                            15 mg     Adsorbs fibrinogen                                          Albumin 28-125 mg Adsorbs antialbumin                                         Histone 48 mg     Substrate for proteolytic enzymes                                             (as 125I histone-Sepharose)                                 Trypsin 30-36 mg  10% activity of unbound enzyme                              Chymo-                                                                        trypsin 45-75 mg  10-25% activity of unbound enzyme                           Lysine  115 μmoles                                                                           Adsorbs 675 CTA units plasminogen                                             per g resin                                                 6-Amino                                                                       Caproic                                                                       Acid    120 μmoles                                                         Benz-                                                                         amidine 150 μmoles                                                                           Adsorbs 15 mg trypsin, 7 mg thrombin                                          per g resin                                                 Heparin 3-6 mg    Adsorbs 3-6 mg antithrombin-III                                               per g resin                                                 ______________________________________                                    

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
 1. In a method for coupling substances selected from the group consisting of protein ligands and non-protein affinity ligands by covalent chemical bonds to insoluble solid supports for the preparation of solid phase catalysts and biospecific adsorbents comprising the steps of reacting the insoluble solid support material with trichloro-s-triazine to produce an activated support and thereafter, coupling the ligand to the activated support, the improvement wherein the production of the activated support is carried out by the steps comprising:(a) reacting a water-free insoluble solid support material with trichloro-s-triazine in a non-aqueous medium comprising an organic solvent which is compatible with the support material and is also a solvent for trichloro-s-triazine; (b) neutralizing the HCl generated during the reaction with a tertiary amine which is soluble in said organic solvent and which does not form an insoluble complex with trichloro-s-triazine in said organic solvent; and (c) washing the obtained dichloro-s-triazine activated support with an additional amount of said organic solvent.
 2. The method of claim 1 wherein the tertiary amine is selected from the group consisting of N,N-diisopropylethylamine and N,N-dimethylaniline.
 3. The method of claim 1 wherein, in step (d) said organic solvent is selected from the group consisting of dioxane and acetonitrile.
 4. The method of claim 1 including the further step of adding a nucleophile to the washed trichloro-s-triazine activated support in an organic solvent to replace one or both of chlorine atoms on the triazine ring, and when both chlorine atoms are replaced, the nucleophile is said ligand.
 5. The method of claim 4 wherein said nucleophile is selected from the group consisting of aliphatic amines, aryl amines, phenols and alkoxyl amines.
 6. The method of claim 1 including the further step of adding a weak nucleophile to the washed dichloro-s-triazine activated support in an organic solvent to replace a single chlorine atom on the triazine ring, leaving the remaining chlorine atom available for further replacement by said ligand.
 7. The method of claim 6 wherein said weak nucleophile is aniline.
 8. The method of claim 6 wherein the monochloro-s-triazine activated support is dried for storage and later coupling with said ligand.
 9. The method of claim 1, wherein said support material initially contains water and is pretreated prior to step (a) to remove said water therefrom.
 10. The method of claim 9 wherein said pretreatment of said support material is accomplished by washing the support material with an organic solvent.
 11. The method of claim 9 wherein said pretreatment of said support material is accomplished by drying the support material under vacuum.
 12. The method of claim 1 wherein said protein ligand is an enzyme.
 13. A support coupled ligand obtained in accordance with the method of claim 1, wherein said non-aqueous medium consists essentially of dioxane or acetonitrile.
 14. A support-coupled ligand obtained in accordance with the method of claim 8, wherein said non-aqueous medium consists essentially of dioxane or acetonitrile.
 15. A support-coupled ligand produced by the method of claim 4, wherein said non-aqueous medium consists essentially of dioxane or acetonitrile.
 16. A support-coupled ligand produced by the method of claim 6, wherein said non-aqueous medium consists essentially of dioxane or acetonitrile. 